This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cotranslational translocation of nascent polypeptides through the Sec61 translocon is an essential step in the processing of secreted membrane proteins. For translating proteins to gain access to the ER lumen, the translocon must first recognize a protein's signal sequence. Previously, our lab has identified certain cyclic heptadepsipeptides, called cotransins, as the first small molecule inhibitors of cotranslational translocation. remarkably, cotransins inhibit translocation in a signal sequence-dependent manner. To date, only ~40 signal sequences have been tested for cotransin sensitivity resulting in few insights into the basis of sensitivity of resistance. Therefore, it is necessary to identify more target signal sequences that are sensitive or resistant to allow for more global comparisons. We intend to identify signal sequence characteristics that determine cotransin sensitivity by using an unbiased proteomics approach. By coupling membrane protein purification techniques and stable isotope labeling in cell culture (SILAC), we will quantify cotransin dependent changes in protein expression to determine the subset of membrane protein signal sequences that are sensitive to cotransin. By comparing sensitive signal sequences to insensitive ones, we will begin to understand the sequence requirements for cotransin sensitivity and uncover the mechanism by which these molecules distinguish between individual signal sequences.